Section 3: Genetics, Cellular and Molecular Biology
Recombinant DNA technology
Subtopic: Fundamentals of Recombinant DNA technology
Question Statement:
A polymerase chain reaction (PCR) was set up with the following reagents: DNA template, Taq
polymerase, buffer, dNTPs, and Mg*. Which one of the following is missing in the reaction
mixture?
Options:
(A) Helicase
(B) Single-stranded binding proteins
(C) Primers
(D) Reverse transcriptase
Official Answer Key:
C
Step-by-Step Solution & Explanation
### **Official Solution & Step-by-Step Derivation (GATE BT 2017 Q.20)**
**1. Governing Concept & Question Classification**:
- **Section**: BT
- **Question Type**: **MCQ** (1 Mark)
- **Topic**: Molecular Biology / Recombinant DNA Technology (Essential Components of PCR)
**2. Detailed Step-by-Step Analysis**:
- A standard Polymerase Chain Reaction (PCR) mixture requires five indispensable components:
1. **DNA template**: Containing the target sequence to be amplified.
2. **DNA Polymerase** (e.g., thermostable *Taq* polymerase): Catalyzes 5′ to 3′ template-directed DNA synthesis.
3. **Reaction Buffer with divalent cations ($\text{Mg}^{2+}$)**: Stabilizes enzyme conformation and catalyzes phosphodiester bond formation.
4. **Deoxynucleotide triphosphates (dNTPs)**: dATP, dCTP, dGTP, dTTP as nucleotide building blocks.
5. **Oligonucleotide Primers**: A pair of synthetic forward and reverse single-stranded DNA primers that provide free 3′-OH groups required for DNA polymerase extension.
- The reagents listed in the question are: *DNA template, Taq polymerase, buffer, dNTPs, and $\text{Mg}^{2+}$.*
- Comparing with the essential components, **primers** are absent from the mixture. Without primers, *Taq* polymerase cannot initiate *de novo* DNA synthesis.
- **Option-by-Option Evaluation**:
- **Option (A) [INCORRECT]**: Helicase is not used in PCR; template denaturation is achieved thermally at $94-98^\circ\text{C}$.
- **Option (B) [INCORRECT]**: Single-stranded binding proteins (SSBs) are not required in standard PCR.
- **Option (C) [CORRECT]**: Primers are missing and strictly indispensable to provide free 3′-OH groups for *Taq* polymerase.
- **Option (D) [INCORRECT]**: Reverse transcriptase is only needed in RT-PCR to transcribe RNA into cDNA, not standard DNA PCR.
**3. Final Answer / Verification Summary**:
- **Official GATE Answer Key**: **C**
- **Scoring Criteria**: +1.0 Mark (Negative marking: -0.33 marks).
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